Fusarium head blight of small grains (“scab”), often referred to by the acronym “FHB”, is increasing world wide and is a tremendous problem for the production and yield of wheat. Within the last dozen years there have been outbreaks of FHB in the midwestern and eastern states in the USA, as well as in central and eastern Canada. The most extended recent episode has been in the spring grain region of the upper midwest, centered on the Red River Valley of North Dakota, Minn., and Manitoba. Here, there have been five consecutive years of severe disease. Losses have been large, and accumulated loss has brought ruin to many farmers.
While several species of the soil- and residue-borne fungus Fusarium are capable of inciting FHB, most of the damage in recent outbreaks in the US and Canada has been due to F. graminearum. In addition to grain crops, this species has a wide host range among grasses. The name Fusarium, in fact, means “of the grasses”. This species was probably present in native grassland long before wheat or barley arrived in North America. F. graminearum is also a superb colonizer of senescent plants; especially corn stalks. F. graminearum is unique in another regard. It is the only common Fusarium species infecting wheat which regularly and abundantly forms its sexual stage (Gibberella zeae) in nature. Because the spores produced by this stage are forcibly shot into the air, they greatly increase the ability of the fungus to disperse from colonized crop residue where the fruit-bodies (perithecia) of this stage form.
F. graminearum has a complex life cycle which is easier to picture if it is divided in two parts: a pathogenic cycle, and a ‘hidden’ cycle of saprophytic colonization. The effect of the pathogenic cycle is seen as FHB. Aerial spores land on flowering heads during wet weather and FHB results. F. graminearum survives on the residue; particularly on infected heads, and sporulates the next spring and summer. In the saprophytic cycle mycelium superficially occupy cornstalks, often without causing disease. At senescence, invasive colonization occurs and F. graminearum takes possession of most of the corn stalk residue. One Minnesota study found over 80% of the corn stalk residue in fall was occupied by F. graminearum and it covers over 60% the following spring. This colonized residue provides a site for massive sporulation during the next growing season. Those airborne spores may begin new saprophytic colonization or they may initiate pathogenic cycles resulting in FHB. The saprophytic colonization cycle is the engine that drives the pathogenic part of the cycle.
The saprophytic life cycle of F. graminearum is fueled by corn stalk residue. There are a number of observations that when corn production moves into a previously small grain area, the occurrence of F. graminearum and the risk of FHB increase. Combine the extension of corn acreage into wheat and barley country with large increases in reduced tillage and the stage is set for major epidemics of FHB in small grains when the weather favoring disease occurs.
Thus Fusarium head blight disease can affect a number of cereal crops such as wheat, barley, rice, rye and maize. It is caused by the phytopathogenic fungi Fusarium graminearum, F. moniliforme, F. culmorum, F. nivale and Microdochium nivale. Moist environmental conditions during anthesis can result in Fusarium epidemics and huge losses in crop revenues. The disease not only reduces crop yield and grain quality but also leads to fungal mycotoxin accumulation in grain.
Kang et al., (Mycol. Res. 104(9): 1083-1093, 2000) disclose experimental evidence that penetration of host tissues by Fusarium culmorum occurred on the inner surfaces of lemma, glume and palea as early as 36 h after inoculation demonstrating that glume, lemma and/or palea are the key entry points for start of the infection process by Fusarium in wheat.
One of the goals of plant genetic engineering is to produce plants with agronomically important characteristics or traits. Recent advances in genetic engineering have provided the requisite tools to transform plants to contain and express foreign genes (Kahl et al., 1995, World Journal of Microbiology and Biotechnology 11:449-460). Particularly desirable traits or qualities of interest for plant genetic engineering would include, but are not limited to, resistance to insects, fungal diseases such as the Fusarium head blight disease, and other pests and disease-causing agents, tolerances to herbicides, enhanced yield stability or shelf-life, environmental tolerances, and nutritional enhancements. The technological advances in plant transformation and regeneration have enabled researchers to take pieces of DNA, such as a gene or genes from a heterologous source, or a native source, but modified to have different or improved qualities, and incorporate the exogenous DNA into the plant's genome. The gene or gene(s) can then be expressed in the plant cell to exhibit the added characteristic(s) or trait(s). In one approach, expression of a novel gene that is not normally expressed in a particular plant or plant tissue may confer a desired phenotypic effect. In another approach, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.
Isolated plant promoters are useful for modifying plants through genetic engineering to have desired phenotypic characteristics. In order to produce such a transgenic plant, a vector that includes a heterologous gene sequence that confers the desired phenotype when expressed in the plant is introduced into the plant cell. The vector also includes a plant promoter that is operably linked to the heterologous gene sequence, often a promoter not normally associated with the heterologous gene. The vector is then introduced into a plant cell to produce a transformed plant cell, and the transformed plant cell is regenerated into a transgenic plant. The promoter controls expression of the introduced DNA sequence to which the promoter is operably linked and thus affects the desired characteristic conferred by the DNA sequence.
Because the promoter is a regulatory element that plays an integral part in the overall expression of a gene or genes, it would be advantageous to have a variety of promoters to tailor gene expression such that a gene or genes is transcribed efficiently at the right time during plant growth and development, in the optimal location in the plant, and in the amount necessary to produce the desired effect. In one case, for example, constitutive expression of a gene product may be beneficial in one location of the plant, but less beneficial in another part of the plant. In other cases, it may be beneficial to have a gene product produced at a certain developmental stage of the plant, or in response to certain environmental or chemical stimuli. The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. It is important when introducing multiple genes into a plant, that each gene is modulated or controlled for optimal expression and that the regulatory elements are diverse, to reduce the potential of gene silencing that can be caused by recombination of homologous sequences. In light of these and other considerations, it is apparent that optimal control of gene expression and regulatory element diversity are important in plant biotechnology.
The proper regulatory sequences must be present in the proper location with respect to the DNA sequence of interest for the newly inserted DNA to be transcribed and thereby, if desired, translated into a protein in the plant cell. These regulatory sequences include, but are not limited to, a promoter, a 5′ untranslated leader, and a 3′ polyadenylation sequence. The ability to select the tissues in which to transcribe such foreign DNA and the time during plant growth in which to obtain transcription of such foreign DNA is also possible through the choice of appropriate promoter sequences that control transcription of these genes.
A variety of different types or classes of promoters can be used for plant genetic engineering. Promoters can be classified on the basis of range of tissue specificity. For example, promoters referred to as constitutive promoters are capable of transcribing operatively linked DNA sequences efficiently and expressing said DNA sequences in multiple tissues. Tissue-enhanced or tissue-specific promoters can be found upstream and operatively linked to DNA sequences normally transcribed in higher levels in certain plant tissues or specifically in certain plant tissues. Other classes of promoters would include, but are not limited to, inducible promoters that can be triggered by external stimuli such as chemical agents, developmental stimuli, or environmental stimuli. Thus, the different types of promoters desired can be obtained by isolating the regulatory regions of DNA sequences that are transcribed and expressed in a constitutive, tissue-enhanced, or inducible manner.
The technological advances of high-throughput sequencing and bioinformatics has provided additional molecular tools for promoter discovery. Particular target plant cells, tissues, or organs at a specific stage of development, or under particular chemical, environmental, or physiological conditions can be used as source material to isolate the mRNA and construct cDNA libraries. The cDNA libraries are quickly sequenced, and the expressed sequences can be catalogued electronically. Using sequence analysis software, thousands of sequences can be analyzed in a short period, and sequences from selected cDNA libraries can be compared. The combination of laboratory and computer-based subtraction methods allows researchers to scan and compare cDNA libraries and identify sequences with a desired expression profile. For example, sequences expressed preferentially in one tissue can be identified by comparing a cDNA library from one tissue to cDNA libraries of other tissues and electronically “subtracting” common sequences to find sequences only expressed in the target tissue of interest. The tissue enhanced sequence can then be used as a probe or primer to clone the corresponding full-length cDNA. A genomic library of the target plant can then be used to isolate the corresponding gene and the associated regulatory elements, including but not limited to promoter sequences.
Despite all the technology currently available no monocotyledonous regulatory sequences capable of regulating transcription of an operably linked nucleic acid sequence in lemma, palea and/or glume monocotyledonous tissue are known. More specifically wheat promoters which could drive expression of a gene in the palea, glume and/or lemma of wheat are unfortunately also unknown. Since the palea, glume and/or lemma are the key entry points susceptible to Fusarium attack, it is highly desirable to have access to specific promoters which can, for instance, drive expression of a heterologous gene able to prevent and/or cure Fusarium attack and/or related disease in these specific tissues.